The decay of mRNA is a key step in eukaryotic gene expression. It is important for early animal development, differentiation, proliferation, the immune response and ensures quality control of gene expression. A critical step in eukaryotic mRNA decay is the removal of the protective 5' terminal cap structure, which precedes and permits degradation of the RNA body by exoribonucleases. The decapping holoenzyme is a ribonucleoprotein assembly containing a catalytic core comprised of Dcp2, activators, and substrate RNA. Dcp1 is an essential interaction partner of Dcp2 that directly enhances enzyme activity. It also functions as a protein interaction platform that allows Dcp1/2 to be recruited to specific transcripts, and promotes cap hydrolysis through a poorly understood mechanism. The Dcp1/2 complex can exist in an ensemble of open and closed states in solution, and prior work suggests conversion to the closed form can impart a switch-like response in activity. Flexibility has encumbered resolution of the active form of the Dcp1/2 complex by crystallography; therefore, in Aim 1 we will determine the structure of the closed, active form of Dcp1/2 bound to non-hydrolyzable cap analogue using an integrated modeling platform that incorporates restraints from structural and biochemical studies. Nonsense-mediated decay (NMD) is a quality control pathway that eliminates transcripts with premature termination codons that would otherwise encode harmful, truncated proteins. Proline-rich nuclear co-receptor 2 (PNRC2), a protein linked to adipogenesis and obesity, promotes decapping of non-sense containing transcripts during NMD and degradation of normal transcripts during Staufen mediated mRNA decay. It functions as an adaptor, linking Dcp1 to trans acting factors that recognize mRNA. PNRC2 also stimulates decapping activity and contains a deeply conserved motif that is required for this effect. In Aim 2 we will study the mechanism of PNRC2 stimulation of decapping during NMD using a combination of in vitro kinetic assays and functional studies in mammalian cells. In Aim 3 we will determine the structural basis for PNRC2 activation of the Dcp1/2 complex using x-ray crystallography in combination with SAXS. These studies will define how adaptors that link Dcp1/2 to specific mRNA decay pathways play a dual role in recruitment and activation. More broadly, the project addresses an emerging paradigm in RNA biology of how a weak, non- specific enzymatic activity can be recruited to a specific mRNA and then activated robustly by associated protein cofactors to ensure correct targeting of substrates.